Recently, various researches for new biomedical technology have been actively progressed by means of the fusion of biotechnology, electronic technology and nano-technology which have been also remarkably developed lately.
Numerous attempts have been made to use patient's cells manipulated in vitro for medical treatment. Various R&D projects for developing new drug for next generation and verifying the drug target by manipulating human cell have been progressed.
The cell manipulation technologies have focused on the development of useful cellular therapeutics. Particularly, various exertions for cell therapy which utilizes the IPS (Induced Pluripotent Stem) cells induced by Yamanaka factor have been tried.
Yamanaka factors refer to four genes, Oct3/4, Sox2, cMyc and K1f4. The insertion of the four genes into the chromosome of a cell by using the vector originated from virus, can transform the somatic cell which already finished differentiation into pluripotent stem cell which can differentiate into various somatic cells. The IPS cell has been evaluated as an innovative technology which can overcome the ethics problem and productivity problem of the embryonic stem cell, and can also obviate the limitations in differentiation capability of the adult stem cell.
However, the IPS cell cause a safety problem that the vector derived from the virus inserted into a live-cell together with the Yamanaka factors. Also, in case of transplantation of the cell or tissue differentiated from the IPS cells which contains the vector derived from the virus into the human body, there may be another problem that tumor risk is increased.
Therefore, new technologies for injecting various materials, such as, DNA, RNA, polypeptide, or nano-particle, directly into a cell without using delivery vehicle, have been required in order to develop new cellular therapeutics which can avoid the above risk caused by using of the viral vector.
In the conventional cell manipulation technologies which do not employ any delivery vehicle, the typical process is to damage the membrane of the cells by mechanical shear force, chemical treatment or by applying electric field and then to allow the material such as genes which exist in extra-cellular fluids to flow into the cell through the damaged membrane gaps, and to expect the damaged cell membrane to be recovered by self-healing capacity of the cell.
A variety of cell transfection techniques, such as particle bombardment, micro-injection and electroporation, have been developed. Except for the micro-injection, these techniques are based on bulk stochastic processes in which cells are transfected randomly by a large number of genes or polypeptides.
The disadvantage of the conventional bulk electroporation the most widely used process for transfection of cells is that the injected dose cannot be controlled.
Therefore, microfluidics-based electroporation has emerged as a new technology for individual cell transfection. The microfluidics-based electroporation offers several important advantages over the bulk electroporation, including lower poration voltages, better transfection efficiency and a sharp reduction in cell mortality.
In 2011, a nanochannel electroporation technology which expose a small area of a cell membrane positioned adjacent to a nanochannel to very large local electric field strength, was disclosed to the public (L. James Lee et al, “Nanochannel electroporation delivers precise amounts of biomolecules into living cells”, Nature Nanotechnology Vol. 6, November 2011, www.nature.com/naturenanotechnology published online on Oct. 16, 2011).
The nanochannel electroporation device comprises two micro channels connected by a nanochannel. The cell to be transfected is positioned in one microchannel to lie against the nanochannel, and other microchannel is filled with the agent to be delivered. The microchannel-nanochannel-microchannel design enables the precise placement of individual cells. One or more voltage pulses lasting milli-seconds is delivered between the two microchannels, causing transfection. Dose control is achieved by adjusting the duration and number of pulses.
By the way, the nanochannel electroporation device disclosed in the above prior art employs PDMS (polydimethylsiloxane) lid that covers microchannel and nanochannels made by polymeric resin through imprinting and formed over the chip substrate.
Therefore, the nanochannel electroporation chip described in the article of Nature Nanotechnology, cannot avoid the chinks occurred between the polydimethylsiloxane lid and the imprinted layer of microchannels and nanochannels made by polymeric resin, because the sealing between the lid and the channel layer of which mechanical properties is different from each other, cannot be absolutely perfect.
Also, since the size stabilities of the polydimethysiloxane lid and the microchannels and nanochannels made by polymeric resin, are low, the sealing between the lid and the layer of channels cannot be perfect. Therefore, the chinks may easily occur between the lid and the layer of channels. The chinks allows the infiltration of the solution which causes the contamination of nanochannel electroporation chip and also generate various aberration of electric field and pressure difference applied for the transfer of cell between the channels and for injection of the transfection agent into the cell.
Therefore, new technology which can put various materials quantitatively into individual cell and can control the amount of the material without such contamination or aberration caused by the contamination has long been anticipated in this technology field.
The inventor of the present application conceived the device for putting material into a cell without using delivery vehicle, formed within one solid material without any sealing in order to exclude the possibility of the occurrence of the chink intrinsically, and thereby, can obviate disadvantages of the prior art, such as the contamination and aberration caused by the chink.
Therefore, the primary object of the present invention is to provide a device for putting material into a cell, formed within one solid and comprises: a first passage on which the cell passes; a second passage on which the material passes and connected to the first passage at a position randomly selected between both ends of the first passage; and an apparatus which applies pressure difference or electric potential difference on the first passage and the second passage.
Another object of the present invention is to provide a process for forming a device for putting material into a cell within one solid by irradiation of LASER, which comprises the steps of: (i) forming a first passage on which the cell passes within the one solid by irradiation of LASER; (ii) forming a second passage on which the material passes and connected the second passage to the first passage at a position randomly selected between both ends of the first passage within the one solid by irradiation of LASER; and (iii) installing an apparatus which applies pressure difference or electric potential difference on the first passage and the second passage.